ES2960206T3 - Mesenchymal stem cells as vaccine adjuvants and methods for using them - Google Patents

Abstract

The present invention provides a method of enhancing an immune response to a vaccine by administering a vaccine and a population of isolated allogeneic human mesenchymal stem cells. The present invention also provides kits comprising a vaccine in a first container and a population of isolated allogeneic human mesenchymal stem cells in a second container. (Automatic translation with Google Translate, without legal value)

Description

DESCRIPTION

Mesenchymal stem cells as vaccine adjuvants and methods for using them

field of invention

The present invention relates to vaccine adjuvants for medical use according to the attached claims. In particular, this invention relates to vaccine adjuvants comprising isolated allogeneic human mesenchymal stem cells that are not genetically manipulated, for medical use in accordance with the appended claims.

Background of the invention

Fragility due to aging and inflammation associated with aging

Frailty due to aging poses a very worrying problem for the health and general well-being of individuals. Frailty due to aging is a geriatric syndrome characterized by weakness, low physical activity, slow motor performance, exhaustion, and involuntary weight loss. See Yao, X. et al., Clinics in Geriatric Medicine 27(1):79-87 (2011). Additionally, there are many studies that show a direct correlation between aging frailty and inflammation. See Hubbard, R.E. et al., Biogerontology 11(5):635-641 (2010).

Immunosenescence is characterized by a chronic low-grade systemic inflammatory state known as aging-associated inflammation. See Franceshi, C. et al., Annals of the New York Academy of Sciences 908:244-254 (2000). This heightened inflammatory state or chronic inflammation found in aging and aging frailty leads to immune dysregulation and complex remodeling of both innate and adaptive immunity. In immunosenescence, the repertoire of T cells and B cells is skewed, resulting in an increase in CD8+ effector memory T cells reexpressing CD45ra (TEMRA) and late/exhausted CD19+ memory B cells, and a decrease in naïve CD8+ T cells, and in switched memory B cells (CD27+). See Blomberg, B.B.et al., Immunologic Research 57(1-3):354-360 (2013); Colonna-Romano, G.et al.,Mechanisms of Aging and Development 130(10):681-690 (2009); and Koch S.et al., Immunity & Aging: 5:6 (2008). This change in the repertoire of T cells and B cells results in a refractory or less efficient immune state. This deterioration of the immune system causes great susceptibility to infectious diseases and a reduction in responses to vaccination. Optimal B cell function is critical for the production of effective antibody responses for vaccines and protection against infectious agents. It is well known that age-associated increases in systemic inflammation (TNF-a, IL-6, IL-8, INFy, and PRC) induce impaired B cell function, leading to poor antibody responses and a decrease in the effectiveness of the vaccine.

Aging-associated inflammation has received considerable attention as it proposes a link between immunological changes and a number of diseases and conditions (such as aging frailty) common in old age. Circulating inflammatory mediators, such as cytokines and acute phase proteins, are markers of low-grade inflammation that have been observed to increase with aging. These proinflammatory cytokines (e.g., TNF-α, IL-6) affect the ability of B cells to produce protective antibodies against exogenous antigens and vaccines. This impaired B cell response is measured by reducing class switch recombination (CCR), which is the ability of immunoglobulins to switch from IgM isotype to a secondary isotype (IgG, IgA, or IgE). Immunoglobulin isotype switching is essential for an adequate immune response, since effector functions differ in each isotype. A key player in RCC and somatic hypermutation (HMS) is the enzyme induced activation cytidine deaminase (IAD), encoded by the geneAicda. The basic function of IAD in RCC and HMS is to initiate breaks in DNA by converting cytosines in uracils in the change and variable regions of immunoglobulins. E47, encoded by the Tcfe2a(E2A) gene, is a transcription factor that belongs to the class I basic helix loop helix (bHBH) proteins, also known as E proteins. Without expression of E47, cell-specific transcription factors B, FBT 1 (early B cell factor) and Pax-5 (paired box protein) are not expressed. Both E47 and Pax-5 are key transcription factors in early B cell lineage development and mature B cell function. See Hagman J.et al., Immunity 27(1):8-10 (2007); Horcher M.et al., Immunity 14(6):779-790 (2001); Riley R.L. et al., Seminars in Immunology 17(5):330-336 (2005). The Pax-5 gene encodes the B cell lineage-specific activating protein (PAEB) that is expressed at all stages of B cell differentiation, but not in terminally differentiated B cells. Pax-5 controls B cell commitment by repressing inappropriate B lineage genes and activating B cell-specific genes, making Pax-5 the gatekeeper of B cells and being expressed exclusively in the B lymphoid lineage since the committed pro-B cell stage to the mature B cell stage. The B cell-specific transcription factor Pax-5 is not only very important in early B cell development and B cell lineage commitment, but is also involved in RCC.

It has also been shown in humans that the amount of TNF-a produced: (1) depends on the amount of inflammation in the system and (2) affects the ability of the B cells themselves to be stimulated with mitogens or antigens. See Frasca, D. et al., Journal of Immunology 188(1):279-286 (2012). Therefore, the immune response in subjects suffering from aging frailty is affected for a number of reasons.

Vaccination in the Elderly

Influenza vaccination is strongly recommended for individuals over 65 years of age to protect them from infection. Although commercially available influenza vaccines provide protection and ensure long-lasting immune memory in children and adults, they are much less effective in older and frail individuals. See Frasca D.et al.,Current Opinion in Immunology 29:112-118 (2014) and Yao X.et al.,Vaccine 29(31):5015-5021 (2011). Despite receiving routine influenza vaccination, elderly individuals are at increased risk of influenza infection leading to secondary complications, hospitalization, physical debilitation, and ultimately death. See Gross, P.et al., Annals of Internal Medicine 123(7):518-527 (1995); Simonsen L. et al., The Journal of Infectious Diseases 178(1):53-60 (1998); and Vu T. et al., Vaccine 20(13-14):1831-1836 (2002). Influenza vaccines also prevent other complications that arise from influenza infection (for example, pneumonia) in most elderly individuals, somewhat reducing the rate of hospitalization. Nichol K.L. et al., The New England Journal of Medicine 331(12):778-784 (1994). However, the hospitalization rate for influenza-related illnesses remains very high. See Thompson, W.W.et al, JAMA 292(11):1333-1340 (2004). Therefore, an improvement in the immune response of a vaccine in the elderly remains necessary.

Previously published results have shown that the specific B cell response to influenza vaccine in vitro (measured by IAD) and the serum response in vivo (measured by HAI assay and ELISA) decrease with aging and are significantly correlated. See Frasca, D. et al., Vaccine 28(51):8077-8084 (2010). It was also found that the percentage of switched memory B cells and CpG-induced IAD, both measured before vaccination (t0), decrease with aging and significantly correlate with the in vivo response. Therefore, these markers are predictive of the live response.

Mesenchymal stem cells

Mesenchymal stem cells are multipotent cells that are capable of migrating to sites of injury, while they are also immunoprivileged by not detectably expressing major histocompatibility complex class II (MHC-II) molecules and expressing MHC-I molecules in low levels. See Le Blanc, K.et al., Lancet 371(9624):1579-1586 (2008) and Klyushnenkova E.etal., J. Biomed. Sci. 12(1):47-57 (2005). As such, allogeneic mesenchymal stem cells hold great promise for therapeutic and regenerative medicine, and have been repeatedly shown to have a high safety and efficacy profile in clinical trials for multiple disease processes. See Hare, J.M.et al.,Journal of the American College of Cardiology 54(24):2277-2286 (2009); Hare, J.M.et al.,Tex. Heart Inst. J. 36(2):145-147 (2009); and Lalu, M.M.et al.,PloS One 7(10):e47559 (2012). It has also been shown that they do not undergo malignant transformation after transplantation into patients. See Togel F.et al.,American Journal of Physiology Renal Physiology 289(1):F31-F42 (2005). Mesenchymal stem cell treatment has been shown to improve severe graft-versus-host disease, protect against acute ischemic renal failure, contribute to the repair of pancreatic and renal glomerular islets in diabetes, reverse fulminant hepatic failure, regenerates damaged lung tissue, attenuates sepsis, and reverses remodeling and improves cardiac function after myocardial infarction. See Le Blanc K.et al.,Lancet 371(9624):1579-1586 (2008); Hare, J.M.et al.,Journal of the American College of Cardiology 54(24):2277-2286 (2009); Togel F.et al.,American Journal of Physiology Renal Physiology 289(1):F31-F42 (2005); Lee R.H. et al., PNAS 103(46):17438-17442 (2006); Parekkadan, B.et al.,PloS One 2(9):e941 (2007); Ishizawa K.et al.,FEBS Letters 556(1-3):249-252 (2004); Nemeth K.et al.,Nature Medicine 15(1):42-49 (2009); Iso Y.et al.,Biochem. Biophys Res. Com.

354(3):700-706 (2007); Schuleri K.H.et al.,Eur. Hearth J. 30(22):2722-2732 (2009); and Heldman A.W.et al, JAMA 311(1):62-73 (2014). Furthermore, mesenchymal stem cells are also a potential source of multiple cell types for use in tissue engineering. See Gong Z.et al.,Methods in Mol. Biol. 698:279-294 (2011); Price, A.P.et al.,Tissue Engineering Part A 16(8):2581-2591 (2010); and Togel F.et al., Organogenesis 7(2):96-100 (2011).

Mesenchymal stem cells have immunomodulatory capacity. They control inflammation and cytokine production by lymphocytes and myeloid-derived immune cells without evidence of immunosuppressive toxicity and are hypoimmunogenic. See Bernardo M.E.et al.,Cell Stem Cell 13(4):392-402 (2013).

Mesenchymal stem cells also have the ability to differentiate not only into cells of mesodermal origin, but also into cells of endodermal and ectodermal origin. See Le Blanc K.et al.,Exp. Hematol.

31(10):890-896 (2003). For example, in vitro, mesenchymal stem cells cultured in airway growth media differentiate to express lung-specific epithelial markers, for example, surfactant protein C, Clara cell secretory protein, and thyroid transcription factor 1. See Jiang Y .et al.,Nature 418(6893):41-49 (2002) and Kotton D.N.et al.,Development 128(24):5181-5188 (2001).

In vivo studies have shown that human mesenchymal stem cells undergo site-specific differentiation into various cell types, including myocytes and cardiomyocytes, when transplanted into sheep fetuses. See Airey J.A.et al., Circulation 109(11):1401-1407 (2004). These mesenchymal stem cells can persist for up to 13 months in multiple tissues after transplantation in non-immunocompromised immunocompetent hosts. Other in vivo studies using rodents, dogs, goats, and baboons similarly demonstrated that human mesenchymal stem cell xenografts do not cause lymphocyte proliferation or systemic alloantibody production in the recipient. See Klyushnenkova E.et al.,J. Biomed. Sci. 12(1):47-57 (2005); Aggarwal S.et al.,Blood 105(4):1815-22 (2005); Augello A.et al.,Arthritis and Rheumatism 56(4):1175-86 (2007); Bartholomew A.et al.,Exp Hematol. 30(1):42-48. (2002); Dokic J.et al., European Journal of Immunology 43(7):1862-72 (2013); Gerdoni E.et al., Annals of Neurology 61(3):219-227 (2007); Lee S.H. etal., Respiratory Research 11:16 (2010); Urban V.S.et al.,Stem Cells 26(1):244-253 (2008); Yang H. et al., PloS One 8(7):e69129 (2013); Zappia E.et al.,Blood 106(5):1755-1761 (2005); Bonfield T.L.et al.,American Journal of Physiology Lung Cellular and Molecular Physiology 299(6):L760-70 (2010); Glenn J.D. et al., World Journal of Stem Cells. 6(5):526-39 (2014); Guo K.et al.,Frontiers in Cell and Developmental Biology 2:8 (2014); Puissant B.et al.,British Journal of Haematology 129(1):118-129 (2005); and Sun L.et al.,Stem Cells 27(6):1421-32 (2009). Taken as a whole, these repeated findings of allogeneic safety and efficacy solidify the notion for using mesenchymal stem cells as an allograft for successful tissue regeneration.

However, despite being a safe therapeutic agent, it is reported in the literature that mesenchymal stem cells exert a suppressive effect on the production of antibodies, as well as on the proliferation and maturation of B cells. See Uccelli, A.et al.,Trends in Immunology 28(5):219-226 (2007). It has also been reported that mesenchymal stem cells inhibit the generation and function of antigen-presenting cells. See Hoogduijn M.J.et al.,Int. Immunopharmacology 10(12):1496-1500 (2010). Finally, it has been reported that mesenchymal stem cells suppress the proliferation of CD4+ and CD8+ T cells. See Ghannam S. et al., Stem Cell Res. & Ther. 1:2 (2010).

Tomchuck S.L. et al, Frontiers in Cellular and Infection Microbiology, Vol 2, 2012, Article 140, reviews the literature identifying the stimulatory effects of mesenchymal stem cells (MSCs) on immune responses and explores the potential of MSCs as a new platform for universal vaccination.

Summary

The invention is established in the set of the attached claims. Embodiments and/or examples of the following description that are not covered by the appended claims are deemed not to be part of the present invention. References to treatment methods in the following paragraphs of this description are to be construed as references to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for the treatment of the human (or animal) body by therapy (or for the diagnosis).

Surprisingly, despite reports that mesenchymal stem cells have a suppressive effect on aspects of the immune system, the present invention provides an adjuvant, wherein the adjuvant is a population of isolated allogeneic human mesenchymal stem cells that are not genetically manipulated. , for use in enhancing the immune response of an elderly human subject to a vaccine or for use in inducing an immune response to a vaccine in an elderly human subject who is a non-responder, wherein the elderly human subject is >65 years of age, and in which immunoprotective amounts of the vaccine and the adjuvant are administered to the subject simultaneously, or sequentially so that the adjuvant is administered before the vaccine.

In one embodiment of the invention, the subject presents symptoms of frailty due to aging. In another embodiment of the invention, the subject presents inflammation associated with aging.

In one embodiment of the invention, the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells. In one embodiment of the invention, the mesenchymal stem cells do not express STRO-1. In another embodiment of the invention, the mesenchymal stem cells do not express CD45. In another embodiment of the invention, the mesenchymal stem cells do not express fibroblast surface markers nor have a fibroblast morphology.

In one embodiment of the invention, the vaccine is monovalent. In another embodiment of the invention, the vaccine is multivalent. In one embodiment of the invention, the vaccine comprises one or more inactivated viruses. In a further embodiment, one or more inactivated viruses are selected from the group consisting of adenovirus, picornavirus, papillomavirus, polyomavirus, hepadnavirus, parvovirus, smallpox virus, Epstein-Barr virus, cytomegalovirus (CMV), herpes virus, roseolovirus , varicella zoster virus, filovirus, paramyxovirus, orthomyxovirus, rhabdovirus, arenavirus, coronavirus, human enterovirus, hepatitis A virus, human rhinovirus, poliovirus, retrovirus, rotavirus, flavivirus, hepacivirus, togavirus and rubella virus. In a further embodiment, the vaccine comprises an inactivated orthomyxovirus. In a further embodiment, the vaccine comprises an inactivated influenza virus. In one embodiment of the invention, the vaccine comprises one or more live attenuated viruses. In a further embodiment, the one or more attenuated viruses are selected from the group consisting of adenovirus, picornavirus, papillomavirus, polyomavirus, hepadnavirus, parvovirus, smallpox virus, Epstein-Barr virus, cytomegalovirus (CMV), herpes virus, roseolovirus, varicella zoster virus, filovirus, paramyxovirus, orthomyxovirus, rhabdovirus, arenavirus, coronavirus, human enterovirus, hepatitis A virus, human rhinovirus, poliovirus, retrovirus, rotavirus, flavivirus, hepacivirus, togavirus and rubella virus

In one embodiment of the invention, the adjuvant is administered at least 1 week before the vaccine is administered. In a further embodiment, the adjuvant is administered at least 2 weeks before the vaccine is administered. In a further embodiment, the adjuvant is administered at least 3 weeks before the vaccine is administered. In a further embodiment, the adjuvant is administered at least 4 weeks before the vaccine is administered. In another embodiment of the invention, the adjuvant is administered at least once a year to improve long-term response to the vaccine.

In one embodiment of the invention, the adjuvant and vaccine are administered parenterally. In one embodiment, the adjuvant is administered systemically. In one embodiment of the invention, the adjuvant is administered by infusion or direct injection. In one embodiment of the invention, the adjuvant is administered intravenously, intra-arterially or intraperitoneally. In a further embodiment, the adjuvant is administered intravenously. In one embodiment of the invention, the vaccine is administered intramuscularly, intravenously, intraarterially, intraperitoneally, subcutaneously, intradermally, orally or intranasally. In a further embodiment, the vaccine is administered intramuscularly.

In one embodiment of the invention, the adjuvant is administered at a dose of approximately 20x106 mesenchymal stem cells. In another embodiment of the invention, the adjuvant is administered at a dose of approximately 100x106 mesenchymal stem cells. In another embodiment of the invention, the adjuvant is administered at a dose of approximately 200x106 mesenchymal stem cells.

In one embodiment of the invention, the mesenchymal stem cells are obtained from a human donor and an HSC matching step of the human donor to the subject prior to administration of the vaccine and adjuvant to the subject is not employed.

In one embodiment of the invention, as a result of the medical use of the adjuvant according to the invention, the expression of intracellular TNF-a in the B cells of the subject decreases at least two-fold compared to the expression levels of TNF-a in B cells from the subject before administration of the adjuvant. In another embodiment of the invention, the ratio of CD4+:CD8+ T cells in the subject increases at least two-fold compared to the ratio of CD4+:CD8+ T cells in the subject prior to administration of the adjuvant. In another embodiment of the invention, the number of switched memory B cells in the subject increases at least two-fold compared to the number of switched memory B cells in the subject before administration of the adjuvant. In another embodiment of the invention, the number of depleted B cells in the subject decreases at least two-fold compared to the number of depleted B cells in the subject prior to administration of the adjuvant.

The adjuvant for use in the invention may be provided as part of a kit having at least two containers, comprising: a vaccine in a first container and the adjuvant in a second container. The mesenchymal stem cells in the kit can be cryopreserved. The kit may further comprise dilution buffer in a third container. The vaccines and mesenchymal stem cells present in the kit may be any of the vaccines or allogeneic human mesenchymal stem cells described herein.

Brief description of the figures

FIG. 1 shows RT-PCR for E47 and GAPDH from five subjects.

FIG. 2 shows IAD mRNA expression correlated with E47 expression. Reproduced from Figure 3 of Frasca, D.et al.,J. Immunol. 180(8):5283-5290 (2008).

FIG. 3 shows the % of changed B cells measured by flow cytometry. An increase in % switch memory B cells was observed in subjects receiving 20x106, 100x106, or 200x106 mesenchymal stem cells 6 months after mesenchymal stem cell infusion compared to the baseline measurement.

Fig. 4 shows the % of switched memory B cells and exhausted B cells, measured by flow cytometry. % switched memory B cells and exhausted B cells were measured in subjects who received a second infusion of mesenchymal stem cells one year after their first infusion of mesenchymal stem cells.

FIG. 5A-5D show T cell concentrations measured by flow cytometry. FIG. 5A-5D show that there is no T cell activation (rejection) induced by allogeneic mesenchymal stem cells at either dose. CD69 is an early marker of T cell activation. CD25 is a late/chronic marker of T cell activation.

FIG. 6 shows CD4+:CD8+ T cell ratios calculated from flow cytometry measurements. An improvement in the CD4+:CD8+ T cell ratio (immune risk phenotype) was observed in subjects who received a second infusion of mesenchymal stem cells one year after their first infusion of mesenchymal stem cells.

FIG. 7A and 7B show the downregulation of intracellular TNF-α in B cells by flow cytometry after infusion of mesenchymal stem cells. "Baseline" refers to flow cytometry measurements taken at baseline. "3 months" refers to flow cytometry measurements taken 3 months after subjects received a second infusion of mesenchymal stem cells one year after their first infusion of mesenchymal stem cells. "FSC-A" is forward scattered light and is proportional to the area or size of the cell surface. "SSC-A" is laterally scattered light and is proportional to cellular granularity or internal complexity. Therefore, correlated measurements of<f>SC-A and SSC-A allow differentiation of cell types in a heterogeneous population (e.g., lymphocytes). "PE-A" is a measure of phycoerythrin, a fluorochrome. "APC-A" is a measure of alphycocyanin, another fluorochrome. FIG. 7A shows flow cytometry measurements of a CRATUS subject. FIG. 7B shows flow cytometry measurements from a second CRATUS subject.

Detailed description

The examples demonstrate that in vivo administration of isolated allogeneic human mesenchymal stem cells that are not genetically manipulated results in an increase in the percentage of switched memory B cells and a decrease in exhausted B cells in the subjects. The examples also demonstrate that in vivo administration of isolated allogeneic human mesenchymal stem cells that are not genetically manipulated results in an improvement in the ratio of CD4+:CD8+ T cells in the subjects. Furthermore, as shown in the examples, intracellular TNF-a is reduced in subjects who have received infusions of allogeneic human mesenchymal stem cells that are not genetically manipulated. From these unexpected results, the present inventors determined that isolated human allogeneic mesenchymal stem cells that are not genetically manipulated are effective in reducing inflammation associated with aging, a predominant characteristic in aging frailty. And because isolated allogeneic human mesenchymal stem cells that are not genetically manipulated have been shown to reduce inflammation associated with aging, these mesenchymal stem cells enhance the immune response to vaccination.

Definitions

The realizations can be practiced without the theoretical aspects presented. On the other hand, the theoretical aspects are presented with the understanding that the realizations are not subject to any theory presented.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by an individual of ordinary skill in the art. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted with a meaning that is consistent with their meaning in the context of the relevant art and should not be interpreted in an idealized or overly formal sense to unless expressly defined herein.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a", and "one, an" and "the, the" are intended to also include the plural forms, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with" or variants thereof are used in the detailed description and/or claims, such terms are intended be inclusive in a way similar to the term “understanding” or “that understands.”

The enumeration of ranges of values herein is simply intended to serve as a shorthand method of referring individually to each separate value that falls within the range, unless otherwise indicated herein, and each separate value is incorporated in the specification as if listed individually herein. The use of any and all of the examples, or exemplary language (e.g., "such as") provided herein, is intended simply to better illuminate the invention and does not represent a limitation on the scope of the invention to unless otherwise claimed. No language in the specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

The term "about" or "approximately" means within an acceptable error range for the particular value determined by an individual of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation, in accordance with practice in the art. Alternatively, "about" may mean a range of ±10% of the reference value.

Dosage, Duration and Subjects

"Immunoprotective amount" means an amount that stimulates a T cell-dependent (DT) immune response. This response is characterized by the ability to elicit significant levels of IgG and opsonic activity. An immunological memory is developed for the immunogenic antigen such that the antibodies produced improve the infection and disease condition mediated by the pathogen and/or prevent infection by the pathogen. The dose and number of doses (e.g., single or multiple doses) administered to the subject will vary depending on a variety of factors, including the route of administration, the conditions and characteristics of the patient (sex, age, body weight, health , size), extent of symptoms, simultaneous treatments, frequency of treatment and desired effect, and the like.

As used herein, the "immunoprotective amount" of a vaccine and adjuvant is determined based on the effect of the combination of the adjuvant and the vaccine. For example, if the adjuvant is shown to significantly improve the immune response of a vaccine, the amount of vaccine needed is less than if the adjuvant improves the immune response of the vaccine less. A person skilled in the art can determine useful amounts of adjuvant and vaccine using known dose development techniques. In one embodiment, the invention comprises a method of enhancing the immune response of a subject to a vaccine or inducing an immune response in a non-responding subject, comprising administering to the subject simultaneously or sequentially the vaccine and an adjuvant in immunoprotective amounts, wherein the adjuvant is a population of isolated allogeneic human mesenchymal stem cells, and further wherein the amount of vaccine required for immunoprotective effect is less than half of that required for immunoprotective effect in the absence of the use of the adjuvant. In other embodiments, the amount of vaccine required for immunoprotective effect is less than 0.1%, less than 0.5%, less than 1.0%, less than 10%, less than 20% or less than 30% of that required for immunoprotective effect in the absence of the use of the adjuvant.

In one embodiment of the invention, the adjuvant is administered simultaneously with the vaccine. In another embodiment of the invention, the adjuvant is administered sequentially with the vaccine such that the adjuvant is administered before the vaccine. In a further embodiment, the adjuvant is administered at least 1 week before the vaccine is administered. In a further embodiment, the adjuvant is administered at least 2 weeks before the vaccine is administered. In a further embodiment, the adjuvant is administered at least 3 weeks before the vaccine is administered. In a further embodiment, the adjuvant is administered at least 4 weeks before the vaccine is administered. In other embodiments, the adjuvant is administered from about 1-8 weeks, 1-12 weeks, 1-36 weeks, 2-8 weeks, 2-12 weeks, 2-26 weeks, 2-36 weeks, 2-48 weeks , 3-4 weeks, 3-12 weeks, 3-8 weeks, 3-26 weeks, 3-36 weeks, 3-48 weeks or 4-12 weeks before vaccine administration. In other embodiments, the adjuvant is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 months before administration of the vaccine, or 1-2 months, 1-3 months, 1-4 months, 1-5 months, 1-6 months, 2-3 months, 2-4 months, 2-6 months or 3 6 months before vaccine administration. In certain embodiments of the invention, the adjuvant is administered at least once a year to improve the long-term response of the subject to the vaccine.

In another embodiment of the invention, the administration of the adjuvant and the vaccine is repeated, such as, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, o 18 months after the first administration of the vaccine, or repeated between 2-4, 2-6, 2-8, 2-10, 3-4, 3-6, 3-8, 3-10, 4-6 , 4-8, 4-10, 6-8, 6-10, 6-12 or 12-18 months after the first administration of the vaccine. Repeated administration of "the vaccine" includes, without limitation, an administration of a vaccine for an influenza virus that is followed by a vaccine for an influenza virus that is not identical to the first vaccine but is recognized as providing vaccination for the type of influenza virus in its prevalent serotypes at the time of the second or repeated administration. In other embodiments, administration of adjuvant and vaccine, simultaneously or sequentially, is repeated three times, four times, five times, six times or 5-10 times. For example, without limitation, the invention includes administering the adjuvant on day 0, then on day 7 administering the vaccine, then on day 180 administering the adjuvant, and then on day 187 administering the vaccine. In certain embodiments, the actual contents of the vaccine may differ during this repeated therapy; however, the vaccine administered at each point of repeat therapy would be directed against the same class of pathogen.

In one embodiment of the invention, the adjuvant is administered at a dose of about 1x106, 2x106, 5x106, 10x106, 20x106, 30x106, 40x106, 50x106, 60x106, 70x106, 80x106, 90x106, 100x106, 110x106, 120x106, 130x106, 140x106 , 150x106, 160x106, 170x106, 180x106, 190x106, 200x106, 300x106, 400x106, 500x106 or 10x107 mesenchymal stem cells. In a further embodiment, the adjuvant is administered at a dose of approximately 20x106 mesenchymal stem cells. In a further embodiment, the adjuvant is administered at a dose of about 100x106 mesenchymal stem cells. In yet a further embodiment, the adjuvant is administered at a dose of about 200x106 mesenchymal stem cells. In additional realizations, the adjuvant is administered in a dose of around 1-400x106, 10-400x106, 100-400x106, 20-200x106, 20-400x106, 0.1-5x106, 0.1-10x106.0.1-100x106, 1-50x106, 1-100x106, 0.01-10x106 or 0.01-100x106 mesenchymal stem cells.

In some embodiments, the immunoprotective amount of adjuvant is sufficient to increase the ratio of CD4+:CD8+ T cells in a subject, such as to increase the ratio of CD4+:CD8+ T cells by at least two, three, four, five, or six-fold. compared to the proportion prior to administration of the adjuvant. In some embodiments, the immunoprotective amount of adjuvant is sufficient to increase the number of switched memory B cells in a subject, such as to increase the number of switched memory B cells by at least two, three, four, or five-fold. compared to the number before administration of the adjuvant. In some embodiments, the immunoprotective amount is sufficient to decrease the expression of intracellular TNF-a in the B cells of a subject, such as to decrease the amounts of intracellular TNF-a in the B cells by at least two, three, four, five or six times compared to the amounts in B cells prior to administration of the adjuvant. In some embodiments, the immunoprotective amount is sufficient to upregulate activation-induced cytidine deaminase (IAD) in a subject. In some embodiments, the immunoprotective amount is sufficient to decrease the number of exhausted B cells in a subject, such as to decrease the number of exhausted B cells at least two to three times compared to the number prior to administration of the adjuvant.

In a further embodiment, the use of an adjuvant allows the administration of lower levels of vaccine than recommended in the absence of adjuvant for the patient population in question and still obtains an immune response. For example, current recommendations call for a single-dose intramuscular injection of 0.5 mL of the high-dose Fluzone vaccine (Sanofi Pasteur) for patients >65 years. In the methods of the present invention, the amount of Fluzone vaccine can be reduced to less than that, such as 0.3 or 0.2 mL for a patient >65 years of age.

The "administration" of a composition can be carried out by oral administration, injection, infusion, parenteral, intravenous, mucosal, sublingual, intramuscular, intradermal, intranasal, intraperitoneal, intraarterial, subcutaneous absorption or by any method in combination with other known techniques. In one embodiment of the invention, the adjuvant is administered systemically. In another embodiment of the invention, the adjuvant is administered by infusion or direct injection. In another embodiment of the invention, the adjuvant is administered intravenously, intra-arterially or intraperitoneally. In a further embodiment, the adjuvant is administered intravenously. In one embodiment of the invention, the vaccine is administered intramuscularly, intravenously, intraarterially, intraperitoneally, subcutaneously, intradermally, orally or intranasally. In a further embodiment, the vaccine is administered intramuscularly.

In some embodiments, the human subject exhibits symptoms of frailty due to aging. In some embodiments, the human subject exhibits inflammation associated with aging.

In one embodiment of the invention, the subject is a non-responder. It is well known that, when a population of individuals is vaccinated against a disease, some of them do not "respond" to the vaccination, that is, their immune system does not seem to react to the administered antigen. This problem is substantial to a greater or lesser degree depending on the diseases and populations involved, but vaccine manufacturers are still trying to reduce, for each of the vaccines they make available to doctors, the number of subjects who are likely to be vaccinated. be "non-responders." This problem is considered to be of particular importance for vaccines comprising purified antigens, such as subunit vaccines produced by genetic engineering.

The term "allogeneic" refers to a cell that is from the same animal species but genetically different at one or more gene loci than the animal that becomes the "recipient host." This usually applies to cells transplanted from one animal to another non-identical animal of the same species.

As used herein, the phrase "in need thereof" means that the subject has been identified as having a need for a particular treatment. In some embodiments, identification may be performed by any diagnostic means. In any of the treatments described herein, the subject may be in need thereof. In some embodiments, the subject is in an environment or will travel to an environment in which a particular disease, disorder or condition is prevalent.

Cells are referred to herein as positive or negative for certain markers. For example, a cell may be negative for CD45, which may also be referred to as CD45-. The superscript notation "-" refers to a cell that is negative for the marker linked to the superscript. In contrast, a marker with a "+" refers to a cell that is positive for that marker. For example, a cell called "CD8+" is positive for CD8. A "+" can also be used to refer to the marker as positive. A "-" can also be used to refer to the marker as negative.

As used herein, the term "stem cell" refers to an embryonic, fetus or adult cell that has, under certain conditions, the ability to reproduce itself for long periods or, in the case of adult stem cells , throughout the life of the organism. It can also give rise to specialized cells that form the body's tissues and organs.

Mesenchymal stem cells are formative pluripotent blast cells that are found, among others, in bone marrow, blood, dermis and periosteum, and that are capable of differentiating into any type of specific mesenchymal or connective tissue (that is, the tissues of the body that support specialized elements, particularly adipose, bone, cartilaginous, elastic and fibrous connective tissues) depending on various influences of bioactive factors, such as cytokines.

Certain methods of isolation and/or purification of mesenchymal stem cells have been described herein and are known in the art. In some embodiments, mesenchymal stem cells are isolated from the bone marrow of adult humans. In some embodiments, cells are passed through a density gradient to eliminate unwanted cell types. Cells can be plated and cultured in appropriate media. In some embodiments, cells are cultured for at least one day or about three to about seven days and non-adherent cells are removed. Adherent cells can then be seeded and expanded.

Other methods of isolating and culturing stem cells are also known. The placenta is an excellent, readily available source of mesenchymal stem cells. Furthermore, mesenchymal stem cells may be derived from adipose tissue and bone marrow stromal cells are speculated to be present in other tissues. While there are dramatic qualitative and quantitative differences in the organs from which adult stem cells can be derived, initial differences between cells may be relatively superficial and balanced by the similar range of plasticity they exhibit.

Homogeneous compositions of human mesenchymal stem cells are provided that serve as progenitors for all mesenchymal cell lineages. Mesenchymal stem cells are identified by specific cell surface markers that are identified with unique monoclonal antibodies. Homogeneous mesenchymal stem cell compositions are obtained by positive selection of adherent or periosteal marrow cells that are free of markers associated with either hematopoietic or differentiated mesenchymal cells. These isolated mesenchymal cell populations display epitopic characteristics uniquely associated with mesenchymal stem cells, have the ability to regenerate in culture without differentiating, and have the ability to differentiate into specific mesenchymal lineages when induced either in vitro or placed in vivo at a site of inflammation.

To obtain human mesenchymal stem cells for use in the invention, the pluripotent mesenchymal stem cells are separated from other cells in the bone marrow or other source of mesenchymal stem cells. Bone marrow cells can be obtained from the iliac crest, femurs, tibias, spine, ribs, or other marrow spaces. Other sites of human mesenchymal stem cells include placenta, umbilical cord, fetal and adolescent skin, and blood.

In some embodiments, human mesenchymal stem cells are identified by the absence of markers. For example, human mesenchymal stem cells useful in the invention include those that are STRO-1 negative and/or CD45 negative. Similarly, human mesenchymal stem cells useful in the invention include those that do not express fibroblast surface markers or have a fibroblast morphology.

Mesenchymal stem cells can be obtained from a human donor and do not employ an MHC matching step from the human donor to the subject prior to administration of the vaccine and adjuvant to the subject.

In one embodiment of the invention, the vaccine is monovalent. In another embodiment of the invention, the vaccine is multivalent.

In one embodiment of the invention, the vaccine comprises one or more inactivated viruses. In a further embodiment, the one or more inactivated viruses are selected from the group consisting of adenovirus, picornavirus, papillomavirus, polyomavirus, hepadnavirus, parvovirus, smallpox virus, Epstein-Barr virus, cytomegalovirus (CMV), herpes virus, roseolovirus, varicella zoster virus, filovirus, paramyxovirus, orthomyxovirus, rhabdovirus, arenavirus, coronavirus, human enterovirus, hepatitis A virus, human rhinovirus, poliovirus, retrovirus, rotavirus, flavivirus, hepacivirus, togavirus and rubella virus. In a further embodiment, the vaccine comprises an inactivated orthomyxovirus. In a further embodiment, the vaccine comprises an inactivated influenza virus.

In one embodiment of the invention, the vaccine comprises one or more live attenuated viruses. In a further embodiment, one or more attenuated viruses are selected from the group consisting of adenovirus, picornavirus, papillomavirus, polyomavirus, hepadnavirus, parvovirus, smallpox virus, Epstein-Barr virus, cytomegalovirus (CMV), herpes virus, roseolovirus , varicella zoster virus, filovirus, paramyxovirus, orthomyxovirus, rhabdovirus, arenavirus, coronavirus, human enterovirus, hepatitis A virus, human rhinovirus, poliovirus, retrovirus, rotavirus, flavivirus, hepacivirus, togavirus and rubella virus.

In another embodiment of the invention, the vaccine comprises an antigen from a bacterial pathogen. In a further embodiment, the bacterial pathogen is selected from the group consisting of: Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio and Yersinia.

In another embodiment of the invention, the vaccine comprises an antigen from a parasitic pathogen. In a further embodiment, the parasitic pathogen is selected from the group consisting of: Acanthamoeba, Anisakis, Ascaris lumbricoides, Balantidium coli, Cestoda, Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania, Linguatula serrate, trematode of liver, Loa boa, Paragonimus, Pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Tapeworm, Toxoplasma gondii, Trypanasoma, Tricocephalus and Wuchereria bancrofti.

In another embodiment of the invention, the vaccine comprises one or more antigenic polypeptides selected from the group consisting of influenza hemagglutinin 1 (HA1), hemagglutinin 2 (HA2), influenza neuraminidase (NA), influenza glycoprotein 1 (gp1). Lassa virus (VLAS), VLAS glycoprotein 2 (gp2), VLAS nucleocapsid-associated protein (PN), VLAS L protein, V<l>AS Z protein, SARS virus S protein, Virus GP2 Ebola, measles virus fusion protein 1 (F 1), HIV-1 transmembrane protein (TM), HIV-1 glycoprotein 41 (gp41), HIV-1 glycoprotein 120 (gp120), HIV-1 glycoprotein 1 (E1 ) hepatitis C virus (HCV) envelope, HCV envelope glycoprotein 2 (E2), HCV nucleocapsid protein (p22), West Nile virus (WNV) envelope glycoprotein (E) , Japanese encephalitis virus (JEV) envelope glycoprotein (E), yellow fever virus (YFV) envelope glycoprotein (E), tick-borne encephalitis virus (E) envelope glycoprotein (VETG), hepatitis G virus (HGV) envelope glycoprotein 1 (E1), respiratory syncytial virus (RSV) fusion protein (F), herpes simplex virus 1 (HSV-1) gD protein, HSV-1 gG protein, HSV-2 gD protein, HSV-2 gG protein, hepatitis B virus (HBV) core protein, Epstein-Barr virus (EBV) glycoprotein 125 (gp125), bacterial outer membrane protein assembly BamA, bacterial protein translocation assembly module TamA, protein-associated polypeptide transporter bacterial protein domain, bacterial surface antigen D15, anthrax protective protein, anthrax lethal factor, anthrax edema factor anthrax, Salmonella typhii S1Da, Salmonella typhii S1Db, cholera toxin, cholera heat shock protein, Clostridium botulinum S antigen, botulinum toxin, Yersinia pestis F1, Yersinia pestis V antigen, Yersinia pestis YopH, Yersinia pestis YopM, Yersinia pestis YopD, Yersinia pestis plasminogen activation (Pla), Plasmodium circumsporozoite protein (PCS), Plasmodium sporozoite surface protein (SSP2/TRAP), Plasmodium liver stage antigen 1 (ATEH), Plasmodium exported protein 1 (EXP 1), binding antigen a Plasmodium erythrocyte 175 (AUE-175), Plasmodium cysteine-rich protective antigen (APRcy), Plasmodium heat shock protein 70 (pct70), Schistosoma m29, and Schistosoma signal transduction protein 14-3-3.

Compositions for use in the invention can be formulated using any suitable method. Formulation of cells with standard pharmaceutically acceptable vehicles and/or excipients can be carried out using routine methods in the pharmaceutical art. The exact nature of a formulation will depend on several factors, including the cells to be administered and the desired route of administration. Suitable formulation types are described in detail in Remington's Pharmaceutical Sciences, 19th edition, Mack Publishing Company, Eastern Pennsylvania, USA.

The compositions may be prepared together with a physiologically acceptable carrier or diluent. Typically, such compositions are prepared as liquid cell suspensions. The cells can be mixed with an excipient that is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, the like and combinations thereof.

Furthermore, if desired, the pharmaceutical compositions of use in the invention may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and/or efficacy-enhancing adjuvants. Such an adjuvant may comprise human serum albumin (HHS).

A suitable carrier or diluent is PlasmaLyte ATM. This is a sterile, non-pyrogenic isotonic solution for intravenous administration. Each 100 mL contains 526 mg of Sodium Chloride, USP (NaCl); 502 mg of Sodium Gluconate (C6HnNaO7); 368 mg Sodium Acetate Trihydrate, USP (C2H3NaO23H2O); 37 mg Potassium Chloride, USP (KCl); and 30 mg of Magnesium Chloride, USP (MgCb6H2O). Does not contain antimicrobial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).

As discussed above, the adjuvant of use in the invention may be provided as part of a kit having at least two containers, comprising a vaccine in a first container and the adjuvant in a second container. Any of the adjuvants or vaccines discussed herein can be formulated in a kit according to the present disclosure. Mesenchymal stem cells can be cryopreserved in the second container. For example, mesenchymal stem cells can be suspended in a cryoprotectant consisting of Hespan® (6% hetastarch in 0.9% sodium chloride) supplemented with 2% ASH and 5% DMSO and subsequently aliquoted into cryopreservation containers. to place them in vapor phase nitrogen freezers. Mesenchymal stem cells can be provided in the second container in PlasmaLyte ATM supplemented with 1% ASH. The kit may have at least three containers, wherein the third container comprises a dilution buffer for suspension and dilution of the mesenchymal stem cells. In such cases, the dilution buffer may contain PlasmaLyte ATM supplemented with 1% ASH.

Examples

Example 1

Effects of aging on E47 mRNA expression in human peripheral blood-derived B cells As discussed above, both E47 and Pax-5 are important transcription factors in early B cell lineage development and B cell function. mature A putative regulatory region in the Aicda gene has been shown to contain binding sites for E47 and Pax-5, both essential for IAD gene expression. The following experiment demonstrates that E47 expression decreases as a function of age and that E47 expression in B cells is positively correlated with IAD expression. See Figures 1 and 2.

Forty-six subjects ranging in age from 20 to 85 years of age were recruited and peripheral blood was drawn. CD19+ B cells (106 cells/mL) were cultured with anti-CD40 (1 pg/mL) and IL-4 (10 ng/mL) for 24 hours. PCR was performed for E47 and GAPDH. E47 was normalized to GAPDH. Figure 1(A) shows undiluted and 1/4 diluted RT-PcR from 5 representative subjects. In Figure 1 (B), the graph shows CT densitometry analyzes normalized to GAPDH. The numbers shown for each sample are percentages of the highest value taken as 100. The Pearson r value for the linear curve expressing the correlation between age and E47 expression is r=-0.84, p=0.00001. The solid line refers to linear regression and the dashed line refers to quadratic regression. Therefore, the data show that E47 expression decreases as a function of age.

Blood from these subjects was also subjected to PCR for IAD and GAPDH, and the results are shown in Figure 2. E47 (from the 24-hour stimulation) and IAD (from the 5-day stimulation) were individually normalized to their value respective of GAPDH. Numbers shown for each sample are percentages of the highest value taken as 100. Data for E47 PCR and IAD were positively correlated. The correlation is significant at the 0.01 level (2-tailed). The solid line refers to linear regression. The expression of E47 in B cells is positively correlated with the expression of IAD as seen in Figure 2. (r=0.80, p level= 0.01, 2-tailed).

Example 2

Memory B cells with change

The following experiment shows that switch memory B cells increase in elderly subjects after treatment with allogeneic mesenchymal stem cells (MSCs). Memory B cells were measured with change at baseline (before intravenous MSC infusion) and 6 months after MSC infusion in human patients from the CRATUS trial. The results show that MSC infusion positively regulates the switched memory B cell compartment, which is a predictive biomarker for enhanced antibody response. See Figure 3. This was true for all 3 MSC doses tested (20x106, 100x106 or 200x106 mesenchymal stem cells). Data show that intravenous administration of allogeneic mesenchymal stem cells doubles B cell switching memory in some patients.

Figure 4 shows the percentage of switched memory B cells and exhausted B cells in subjects who received a second infusion of mesenchymal stem cells one year after their first infusion of mesenchymal stem cells. The graphs suggest that a second injection at 12 months may be necessary to maintain the improvements in immune cells derived from the initial mesenchymal stem cell treatment.

Example 3

T Cell Activation

The following experiment shows that both early and late/chronic T cell activation decreases after allogeneic MSC treatment. The early marker of T cell activation (CD69) and the late/chronic marker of T cell activation (CD25) were measured in human patients from the CRATUS test sample. As shown in Figures 5A and 5B, there is no T cell activation (rejection) induced by allogeneic mesenchymal stem cells at any dose (20x106, 100x106 or 200x106 mesenchymal stem cells). See also Figures 5C and 5D, which show the absolute difference six months after MSC infusion versus the percentage of CD69 or CD25 cells.

Example 4

Immune Risk Phenotype

The immune risk phenotype was measured in the same CRATUS subjects who underwent a second MSC infusion one year after the first MSC infusion. See Figure 6. Results show that MSC infusion improves the ratio of CD4+:CD8+ T cells. Furthermore, 12 months after the first infusion, the effects begin to recede. At that point, a second MSC infusion produces additional improvement. A second injection may be necessary at 12 months to maintain the improvements in immune cells derived from the initial mesenchymal stem cell treatment.

Example 5

FNT-Alpha

TNF-a reduces IAD. TNF-a was measured in samples from two CRATUS subjects by intracellular staining of B cells by flow cytometry and confirmed by qPCR. Results are shown at baseline (12 months after the first MSC infusion) and three months (three months after the second infusion) as shown in Figures 7A and 7B. Results show downregulation of intracellular TNF-α in B cells by flow cytometry after infusion of mesenchymal stem cells. These results were confirmed by qPCR.

Example 6

Effects of Intravenous Administration of Allogeneic Human Mesenchymal Stem Cells on Vaccine-Specific Antibody Responses in Patients with Frailty of Aging: the HRAE Trial (Phase I/II)

The HRAE trial is a phase I/II, randomized, double-blind, placebo-controlled study. The main objective of the study is to demonstrate that intravenous administration of mesenchymal stem cells can improve adaptive immunity and can improve the primary B cell response to influenza vaccine in subjects with aging frailty.

Forty-three (43) subjects with frailty due to aging were enrolled. The adjuvant (allogeneic mesenchymal stem cells) was administered by peripheral intravenous infusion. The total duration for each subject after infusion is 12 months, plus up to an additional 2 months for Screening and Baseline visits. A Safety Encounter is followed by a double-blind randomized phase. All subjects must meet inclusion/exclusion criteria and are evaluated before scheduled infusion to establish baseline.

Security Meeting

The Safety Meeting includes 23 subjects in 3 cohorts and is conducted to determine the optimal timing for administering influenza vaccine after mesenchymal stem cell infusion.

Cohort A (3 subjects): A single peripheral intravenous infusion of 20x106 mesenchymal stem cells is administered to each subject. At 1 week after infusion, subjects receive an intramuscular injection of a single 0.5 mL dose of the high-dose Fluzone vaccine (Sanofi Pasteur), which is recommended for patients >65 years of age. These subjects are infused first (i.e., before any subjects in Cohorts B and C) and are infused no less than 5 days apart.

Cohort B (10 subjects): A single peripheral intravenous infusion of 100x106 mesenchymal stem cells is administered to each subject. At 1 week post-infusion, subjects receive a single-dose intramuscular injection of 0.5 mL of high-dose Fluzone vaccine.

Cohort C (10 subjects): A single peripheral intravenous infusion of 100x106 mesenchymal stem cells is administered to each subject. At 4 weeks post-infusion, subjects receive a single-dose intramuscular injection of 0.5 mL of the high-dose Fluzone vaccine.

Subjects in Cohorts B and C are randomized, and the first 3 subjects are infused no less than 5 days apart. Follow-up Visits will occur 1 and 4 weeks after vaccination to determine the safety and efficacy of vaccination after mesenchymal stem cell infusion. After all 23 subjects have been infused and vaccinated, a 30-day review is performed to evaluate all safety data. The Double-Blind Randomized Phase is conducted after successful completion of the Safety Encounter has been reviewed and approved. After the Safety Meeting data have been analyzed, both 1 week and 4 weeks after vaccination, a standard vaccination time point is used for the Double-Blind Randomized portion of the trial.

Double-Blind Randomized Trial

The Double-Blind Randomized Trial includes 20 subjects in 2 cohorts. All subjects must meet inclusion/exclusion criteria and be evaluated prior to scheduled infusion to establish baseline. Subjects are randomized in a 1:1 ratio into 2 cohorts as follows:

Cohort 1 (10 subjects): A single peripheral intravenous infusion of 100x106 mesenchymal stem cells is administered to each subject. At the optimal post-infusion time point (1 or 4 weeks, as determined at the Safety Meeting), subjects receive an intramuscular injection of a single 0.5 mL dose of the high-dose Fluzone vaccine.

Cohort 2 (10 subjects): A single intravenous infusion of placebo (PlasmaLyte ATM with 1% ASH) is administered to each subject. At the optimal post-infusion time point (1 or 4 weeks, as determined at the Safety Meeting), subjects receive an intramuscular injection of a single 0.5 mL dose of the high-dose Fluzone vaccine.

Follow-up is done by phone call 1 day after infusion and after vaccination. Follow-up in-office visits occur at Week 1 and Week 4 post-vaccination, and at Month 6 and Month 12 post-infusion, to complete all safety and efficacy assessments. Any subject who develops influenza-like symptoms during the first 7 months after vaccination should immediately schedule an office visit so that the possible influenza strain can be evaluated.

Primary Endpoint

The primary efficacy endpoint is B cell function as measured by (1) the ability of B cells to upregulate activation-induced cytidine deaminase (IAD) in response to CpG or influenza vaccine via qPCR and (2) the production of influenza-specific antibodies through HAI and ELISA. Data for the primary efficacy endpoint are obtained from the Baseline Visit, the Vaccination Visit (conducted 1 or 4 weeks after the mesenchymal stem cell infusion), the Follow-up Visits after vaccination of the Week 1 and Week 4 and Month 6 and Month 12 Follow-up Visits.

Inclusion criteria

All subjects enrolled in this trial must: provide written informed consent; be between 65 and 95 years of age at the time of signing the Informed Consent Form; have a diagnosis of frailty, with a score of 4 to 7 according to the Canadian Frailty Scale; show immunosenescence measured by flow cytometry staining of whole blood, resulting in <5% switched memory B cells, delayed/exhausted memory >10%, naïve CD8+ cells <20 }%, and TEMRA CD8+ cells >40%; and have total bilirubin between 0.3 and 1.9 mg/dL.

Adjuvant and Placebo

The final mesenchymal stem cell formulation is 2.5x106 mesenchymal stem cells/mL suspended in 80 mL of PlasmaLyte ATM containing 1.0% ASH so that subjects receive 100 x106 mesenchymal stem cells. For subjects in the Encounter Phase receiving 20 x106 mesenchymal stem cells, the final formulation is 0.5 x106 mesenchymal stem cells/mL suspended in 80 mL of PlasmaLyte ATM containing 1.0% ASH. The adjuvant is manufactured by the Cell Processing Facility of Longeveron LLC (Life Sciences and Technology Park, 1951 NW 7th Ave., Miami, FL 33136).

The dilution buffer is used as a placebo. The final placebo formulation is PlasmaLyte ATM containing 1.0% ASH (80 mL total).

Adjuvant dosage and administration rate

Subjects in this trial receive a single infusion of 20x106 mesenchymal stem cells, 100x106 mesenchymal stem cells, or placebo. The maximum infusion rate is 2.5x106 mesenchymal stem cells/min, which is well below the maximum reported dosing rate. Table 3 provides a breakdown of infusion parameters for placebo and mesenchymal stem cell infusions. A total of 80 mL is administered intravenously to each subject.

Claims (14)

1. An adjuvant, wherein the adjuvant is a population of isolated allogeneic human mesenchymal stem cells that are not genetically manipulated, for use in enhancing the immune response of an elderly human subject to a vaccine or for use in inducing a response immune to a vaccine in a non-responding elderly human subject, in which the elderly human subject is >65 years of age, and wherein immunoprotective amounts of the vaccine and the adjuvant are administered to the subject simultaneously or sequentially such that the adjuvant is administered before the vaccine. 2. The adjuvant for use as claimed in claim 1, wherein the human subject exhibits symptoms of frailty due to aging. 3. The adjuvant for use as claimed in claim 1 or claim 2, wherein the human subject exhibits inflammation associated with aging. 4. The adjuvant for use as claimed in any one of claims 1-3, wherein: (i) mesenchymal stem cells do not express STRO-1; I (ii) mesenchymal stem cells do not express CD45; I (iii) mesenchymal stem cells do not express fibroblast surface markers nor have a fibroblast morphology. 5. The adjuvant for use as claimed in any one of claims 1 to 4, wherein the vaccine is multivalent. 6. The adjuvant for use as claimed in any one of claims 1 to 5, wherein the vaccine comprises one or more inactivated viruses, preferably wherein one or more inactivated viruses are selected from the group consisting of adenovirus, picornavirus , papillomavirus, polyomavirus, hepadnavirus, parvovirus, smallpox virus, Epstein-Barr virus, cytomegalovirus (CMV), herpes virus, roseolovirus, varicella zoster virus, filovirus, paramyxovirus, orthomyxovirus, rhabdovirus, arenavirus, coronavirus, human enterovirus , hepatitis A virus, human rhinovirus, poliovirus, retrovirus, rotavirus, flavivirus, hepacivirus, togavirus and rubella virus. 7. The adjuvant for use as claimed in claim 6, wherein the vaccine comprises an inactivated orthomyxovirus. 8. The adjuvant for use as claimed in claim 7, wherein the vaccine comprises an inactivated influenza virus. 9. The adjuvant for use as claimed in any one of claims 1 to 5, wherein the vaccine comprises one or more live attenuated viruses, preferably wherein one or more attenuated viruses are selected from the group consisting of adenoviruses, picornavirus, papillomavirus, polyomavirus, hepadnavirus, parvovirus, smallpox virus, Epstein-Barr virus, cytomegalovirus (CMV), herpes virus, roseolovirus, varicella zoster virus, filovirus, paramyxovirus, orthomyxovirus, rhabdovirus, arenavirus, coronavirus, enterovirus human, hepatitis A virus, human rhinovirus, poliovirus, retrovirus, rotavirus, flavivirus, hepacivirus, togavirus and rubella virus. 10. The adjuvant for use as claimed in any one of claims 1-9, wherein: (i) the adjuvant is administered at least 1 week before the vaccine is administered; (ii) the adjuvant is administered at least 2 weeks before the vaccine is administered; (iii) the adjuvant is administered at least 3 weeks before the vaccine is administered; (iv) the adjuvant is administered at least 4 weeks before the vaccine is administered; either (v) the adjuvant is administered at least once a year to improve long-term response to the vaccine. 11. The adjuvant for use as claimed in any one of claims 1-10, wherein: (i) the adjuvant is administered at a dose of about 20x106 mesenchymal stem cells; either (ii) the adjuvant is administered at a dose of about 100x106 mesenchymal stem cells; either (Ni) adjuvant is administered at a dose of around 200x106 mesenchymal stem cells. 12. The adjuvant for use as claimed in any one of claims 1-11, wherein the mesenchymal stem cells are obtained from a human donor and wherein an MHC matching step of the human donor with the subject prior to administration of the vaccine and adjuvant to the human subject. 13. The adjuvant for use as claimed in any one of claims 1-12, further comprising repeated administration of the vaccine and the adjuvant at least six months after the first administration of the vaccine. 14. The adjuvant for use as claimed in claim 1, wherein: (i) the intracellular expression of TNF-a in B cells of the subject decreases at least two-fold compared to the expression levels of TNF-a in B cells of the subject before administration of the adjuvant; either (ii) the ratio of CD4+:CD8+ T cells in the subject increases at least two-fold compared to the ratio of CD4+:CD8+ T cells in the subject before administration of the adjuvant; either (iii) the number of switched memory B cells in the subject increases at least two-fold compared to the number of switched memory B cells in the subject before administration of the adjuvant; either (iv) the number of exhausted B cells in the subject decreases at least two-fold compared to the number of exhausted B cells in the subject before administration of the adjuvant.